Assessment of the vaginal residence time of biomarkers of semen exposure.

OBJECTIVE: The primary objective of this pilot study is to determine and compare the residence time in the vagina of biomarkers of semen exposure for up to 15 days post exposure. The biomarkers are prostate-specific antigen (PSA), Y chromosome DNA, the sex determining region of the Y chromosome (SRY) and testis-specific protein Y-encoded 4 (TSPY4). The secondary objectives are to determine if biomarker concentrations differed between intercourse and inoculation groups, to establish whether the sampling frequency post exposure affected biomarker concentrations and decay profile and to determine if biomarker concentrations in vaginal swabs obtained by the participant at home were similar to swabs obtained by the nurse in the clinic. STUDY DESIGN: We randomized healthy women to unprotected intercourse (n=17) versus vaginal inoculation with the male partner's semen in the clinic (n=16). Women were then further randomized to have vaginal swabs obtained at either 7 or 4 time points after semen exposure, up to 15 days post exposure, either obtained at home by the participant or in the clinic by the research nurse. RESULTS: PSA and SRY were markers of recent semen exposure. TSPY4 was detectable in approximately 50% of participants at 15 days post exposure. Unprotected intercourse resulted in significantly higher concentrations of select biomarkers. Sampling frequency and home versus clinic sampling had no significant effect on biomarker concentrations. CONCLUSIONS: Objective biomarkers of recent or distant semen exposure may have great utility for verifying protocol compliance in a variety of clinical trials.

Label-free detection of sex determining region Y (SRY) via capacitive biosensor.

In this work, we present for the first time, the use of a simple fractal capacitive biosensor for the quantification and detection of sex-determining region Y (SRY) genes. This section of genetic code, which is found on the Y chromosome, finds importance for study as it causes fetuses to develop characteristics of male sex-like gonads when a mutation occurs. It is also an important genetic code in men, and disorders involving the SRY gene can cause infertility and sexual malfunction that lead to a variety of gene mutational disorders. We have therefore designed silicon-based, label-free fractal capacitive biosensors to quantify various proteins and genes. We take advantage of a good dielectric material, Parylene C for enhancing the performance of the sensors. We have integrated these sensors with a simple microchannel for easy handling of fluids on the detection area. The read-out value of an Agilent LCR meter used to measure capacitance of the sensor at a frequency of 1 MHz determined gene specificity and gene quantification. These data revealed that the capacitance measurement of the capacitive biosensor for the SRY gene depended on both the target and the concentration of DNA. The experimental outcomes in the present study can be used to detect DNA and its variations in crucial fields that have a great impact on our daily lives, such as clinical and veterinary diagnostics, industrial and environmental testing and forensic sciences.

A Clinical Case of an SRY-Positive Intersex/Hermaphrodite Holstein Cattle.

A single-born, 15-month-old Holstein cattle, diagnosed as hermaphrodite, was investigated for estrous cycle, hormonal profiles, karyotype, presence of SRY, as well as anatomopathological and histological aspects. Normal continuous estrous cycles and basal testosterone levels were reported. Necropsy showed the presence of a female genital tract that mismatched a vulvar opening and a male pelvic urethra continued within a penis. Moreover, we observed islands of seminiferous tubules with the presence of germline cells, 2 pampiniform plexi, the corpus cavernosum, the penile urethra, the corpus spongiosum and the glans. Cytogenetic analyses of the blood cells showed an XX karyotype, while the molecular analyses revealed the presence of the SRY gene in several tissues, including blood. This is the first report in the scientific literature of an SRY-positive hermaphrodite Holstein cattle with continuous ovarian cycles.

Bilateral Ovotestes in a 78, XX SRY-Negative Beagle Dog.

This report describes a disorder of the sexual development in a beagle dog resulting in an intersex condition. A 6 mo old beagle was presented for evaluation of a protruding structure from the vulva consistent with an enlarged clitoris. Ultrasonographic examination revealed the presence of both gonadal and uterine structures. Retrograde cystourethrovaginogram showed the presence of an os clitoris and severe vaginal stenosis. Histological studies revealed the presence of bilateral ovotestes and uterus. The gonad had interstitial cells within seminiferous-like tubules lined only with Sertoli cells and abundant interstitial cells among primordial, primary, and secondary follicles. Hormone assays completed before and after gonadohysterectomy showed an elevation in the levels of progesterone and dihydrotestosterone that returned to baseline 3 mo after surgery. Testosterone levels that were within the male reference ranges before surgery decreased to basal levels postsurgically. 17-ß-Estradiol levels showed little variation and values were always within the reference ranges for a male. Cytogenetic analysis showed a normal female karyotype (2n = 78, XX) and polymerase chain reaction analysis revealed the absence of the sex-determining region Y gene. In summary, the dog presented bilateral ovotestes and a 2n = 78, XX chromosomal complement lacking the sex determining region Y gene, consistent with a diagnosis of true hermaphroditism.

TSPY4 is a novel sperm-specific biomarker of semen exposure in human cervicovaginal fluids; potential use in HIV prevention and contraception studies.

BACKGROUND: Developing an objective, reliable method to determine semen exposure in cervicovaginal fluids is important for accurately studying the efficacy of vaginal microbicides and contraceptives. Y-chromosome biomarkers offer better stability, sensitivity, and specificity than protein biomarkers. TSPY4 belongs to the TSPY (testis-specific protein Y-encoded) family of homologous genes on the Y-chromosome. Using a multiplex PCR amplifying TSPY4, amelogenin, and Sex-determining region in the Y chromosome (SRY), our objective was to determine whether a gene in the TSPY family was a more sensitive marker of semen exposure in cervicovaginal fluids than SRY. STUDY DESIGN: The multiplex polymerase chain reaction (PCR) was developed using sperm and vaginal epithelial (female) DNA. Diluted sperm DNA and mixed male/female DNA was used to determine the sensitivity of the multiplex PCR. Potential interference of TSPY4 amplification by components in cervicovaginal and seminal fluids was determined. TSPY4 and SRY amplification was also investigated in women participating in a separate IRB-approved clinical study in which cervicovaginal swab DNA was collected before semen exposure and at various time points after exposure. RESULTS: TSPY4, SRY, and amelogenin were amplified in sperm DNA, but only amelogenin in female DNA. The limit of sperm DNA from which TSPY4 could be amplified was lower than SRY (4 pg vs 80 pg). TSPY4 could also be amplified from mixed male/female DNA. Amplification was not affected by cervicovaginal and seminal components. Using cervicovaginal swab DNA from three women before and after semen exposure, TSPY4 was detected up to 72 h post exposure while SRY detection was observed up to 24-48 h. TSPY4 was detected up to 7 days post exposure in one out of three women. CONCLUSIONS: We have demonstrated that TSPY4 is a new sensitive, and sperm-specific biomarker. The multiplex PCR incorporating this new biomarker has potential to be an objective measure for determining semen exposure in clinical trials of vaginal products such as contraceptives and HIV pre/post-exposure prophylaxis agents.

Quantitative analysis of fetal DNA in maternal plasma in gestational diabetes mellitus, iron deficiency anemia and gestational hypertension pregnancies.

OBJECTIVE: To quantify circulating fetal DNA (fDNA) levels in the second and third trimesters of normal healthy pregnant individuals and pregnant women with the following clinical conditions: gestational diabetes mellitus (GDM), iron deficiency anemia and gestational hypertension (GHT). METHODS: The SRY gene located on the Y chromosome was used as a unique fetal marker. The fDNA was extracted from maternal plasma and the SRY gene concentrations were measured by quantitative real-time polymerase chain reaction (PCR) amplification using TaqMan dual labeled probe system. RESULTS: No significant differences were observed in the mean fDNA concentration between normal and GDM pregnancy samples (p > 0.05) and also between normal and anemic pregnancy samples (p > 0.05) in both trimesters, but significant differences were observed between the third trimester normal and GHT pregnancy samples (p = 0.001). GDM and iron deficiency anemia do not affect the levels of fDNA in maternal plasma while GHT significantly elevates the levels of fDNA in maternal plasma. CONCLUSIONS: Increased amount of circulating fDNA in maternal plasma could be used for early identification of adverse pregnancies. GDM and anemia do not affect the levels of fDNA in maternal plasma while GHT significantly elevates the levels of fDNA in maternal plasma. Hence, the elevated fDNA values could be used as a potential screening marker in pregnancies complicated with GHT but not with GDM and iron deficiency anemia.

GTG mutation in the start codon of the androgen receptor gene in a family of horses with 64,XY disorder of sex development.

Genetic sex in mammals is determined by the sex chromosomal composition of the zygote. The X and Y chromosomes are responsible for numerous factors that must work in close concert for the proper development of a healthy sexual phenotype. The role of androgens in case of XY chromosomal constitution is crucial for normal male sex differentiation. The intracellular androgenic action is mediated by the androgen receptor (AR), and its impaired function leads to a myriad of syndromes with severe clinical consequences, most notably androgen insensitivity syndrome and prostate cancer. In this paper, we investigated the possibility that an alteration of the equine AR gene explains a recently described familial XY, SRY + disorder of sex development. We uncovered a transition in the first nucleotide of the AR start codon (c.1A>G). To our knowledge, this represents the first causative AR mutation described in domestic animals. It is also a rarely observed mutation in eukaryotes and is unique among the >750 entries of the human androgen receptor mutation database. In addition, we found another quiet missense mutation in exon 1 (c.322C>T). Transcription of AR was confirmed by RT-PCR amplification of several exons. Translation of the full-length AR protein from the initiating GTG start codon was confirmed by Western blot using N- and C-terminal-specific antibodies. Two smaller peptides (25 and 14 amino acids long) were identified from the middle of exon 1 and across exons 5 and 6 by mass spectrometry. Based upon our experimental data and the supporting literature, it appears that the AR is expressed as a full-length protein and in a functional form, and the observed phenotype is the result of reduced AR protein expression levels.

Hypospadias in a male (78,XY; SRY-positive) dog and sex reversal female (78,XX; SRY-negative) dogs: clinical, histological and genetic studies.

Hypospadias is rarely reported in dogs. In this study we pre-sent 2 novel cases of this disorder of sexual development and, in addition, a case of hereditary sex reversal in a female with an enlarged clitoris. The first case was a male Moscow watchdog with a normal karyotype (78,XY) and the presence of the SRY gene. In this dog, perineal hypospadias, bilateral inguinal cryptorchidism and testes were observed. The second case, representing the Cocker spaniel breed, had a small penis with a hypospadic orifice of the urethra, bilateral cryptorchidism, testis and a rudimentary gonad inside an ovarian bursa, a normal female karyotype (78,XX) and a lack of the SRY gene. This animal was classified as a compound sex reversal (78,XX, SRY-negative) with the hypospadias syndrome. The third case was a Cocker spaniel female with an enlarged clitoris and internally located ovotestes. Cytogenetic and molecular analyses revealed a normal female karyotype (78,XX) and a lack of the SRY gene, while histology of the gonads showed an ovotesticular structure. This case was classified as a typical hereditary sex reversal syndrome (78,XX, SRY-negative). Molecular studies were focused on coding sequences of the SRY gene (case 1) and 2 candidates for monogenic hypospadias, namely MAMLD1 (mastermind-like domain containing 1) and SRD5A2 (steroid-5-alpha-reductase, alpha polypeptide 2). Sequencing of the entire SRY gene, including 5'- and 3'-flanking regions, did not reveal any mutation. The entire coding sequence of MAMLD1 and SRD5A2 was analyzed in all the intersexes, as well as in 4 phenotypically normal control dogs (3 females and 1 male). In MAMLD1 2 SNPs, including 1 missense substitution in exon 1 (c.128A>G, Asp43Ser), were identified, whereas in SRD5A2 7 polymorphisms, including 1 missense SNP (c.358G>A, Ala120Thr), were found. None of the identified polymorphisms cosegregated with the intersexual phenotype, thus, we cannot confirm that hypospadias may be associated with polymorphism in the coding sequence of the studied genes.

Disorder of sex development (XX male, SRY negative) in a French bulldog.

A female French bulldog was presented with an enlarged clitoris. Abdominal surgery revealed a normal uterus and gonads resembling testes. Histologically, the gonads contained seminiferous tubules. The karyotype was XX, and the SRY gene was not detected. A diagnosis of XX male, SRY negative disorder of sexual development was made.

Late transplantation of allogeneic bone marrow stromal cells improves neurologic deficits subsequent to intracerebral hemorrhage.

BACKGROUND AIMS: Stem cell therapy seems to be a promising therapeutic tool for treating central nervous system (CNS) injuries. Bone marrow stromal cell (BMSC) transplantation influences functional outcome subsequent to intracerebral hemorrhage (ICH), and enhances endogenous neurogenesis in acute condition studies. We investigated whether late administration of BMSC improves functional deficits subsequent to ICH. METHODS: Experimental ICH was induced by stereotactic injection of 0.5 IU collagenase type IV in the striatum of adult female Wistar rats, and 2 months later intralesional administration of 5 × 10(6) allogeneic BMSC from male donors rats in saline (n = 10), or saline only (n = 10), was performed. In the following 6 months, functional outcome was evaluated in each animal by rotarod, modified neurologic severity score (mNSS) and video-tracking box (VTB) tests. To study the behavior of BMSC after transplantation, in situ hybridization studies were performed, with double labeling of the chromosome Y-linked SrY-gene, and neuronal nuclei (NeuN) protein or gliofibrillary acidic protein (GFAP). RESULTS: The assessment test revealed significant improvements in functional outcome for the BMSC-treated animals after 2 months of follow-up. Histologic results showed that functional outcome was associated with strong reactivation of endogenous neurogenesis. Furthermore, intralesional BMSC not only integrated in the injured tissue but also showed phenotypic expression of GFAP and NeuN. CONCLUSIONS: Late intracerebral transplantation of allogeneic BMSC induces functional recovery after ICH. The possibility of using this type of cell therapy to reverse the consequences of hemorrhagic stroke in humans should be considered.

Sry-negative XX sex reversal in a French Bulldog.

Here, we describe a 3-month-old XX male French Bulldog. The diagnosis was based on the clinical signs, gonadal histology and cytogenetic analysis. Additionally, the dog was confirmed to be Sry negative by semi-quantitative reverse transcription polymerase chain reaction (sqRT-PCR). Canine Sry-negative XX sex reversal is a disorder of gonadal development where individuals who have a female karyotype develop testes or ovotestes. To our knowledge, this case is the first XX male sex reversion described in a French Bulldog.

SRY interacts with ribosomal proteins S7 and L13a in nuclear speckles.

The SRY (sex-determining region on the Y chromosome) is essential for male development; however, the molecular mechanism by which the SRY induces testis development is still unclear. To elucidate the mechanism of testis development, we identified SRY-interacting proteins using a yeast two-hybrid system. We found two ribosomal proteins, RPS7 (ribosomal protein S7) and RPL13a (ribosomal protein L13a) that interact with the HMG (high-mobility group) box domain of SRY. Furthermore, we confirmed the intracellular distributions of RPS7, RPL13a and SRY and found that the three proteins were co-expressed in COS1 cells. SRY, RPS7 and RPL13a were co-localized in nuclear speckles. These findings suggest that SRY plays an important role in activities associated with nuclear speckles via an unknown mechanism.

Fetal sex determination using circulating cell-free fetal DNA (ccffDNA) at 11 to 13 weeks of gestation.

OBJECTIVE: To examine the performance of a mass spectrometry-based detection platform using three Y-chromosome sequences for fetal sex determination from circulating cell-free fetal DNA (ccffDNA) in maternal blood in the first trimester of pregnancy. METHODS: We extracted ccffDNA for the determination of fetal sex from stored maternal plasma obtained at 11 to 13 weeks' gestation from singleton pregnancies with documented fetal gender. Mass spectrometry was used to examine 236 specimens for the presence of three Y-chromosome sequences (SRY, DBY and TTTY2). The sample was classified as male, female or inconclusive depending on the detection of three, one/none and two sequences, respectively. RESULTS: Three (1.3%) of the 236 cases were classified as invalid due to the absence of a well-defined spectral peak for TGIF and 22 (9.3%) were reported as inconclusive. In the 211 cases with a valid result, the fetal sex was correctly identified in 90 of 91 male babies and 119 of 120 female babies giving an accuracy of 99.1% and sensitivity and specificity for prediction of male fetuses of 98.9 and 99.2%, respectively. CONCLUSION: Fetal sex determination can be accurately determined from maternal ccffDNA in the first trimester of pregnancy using mass spectrometry analysis.

Minimal external masculinization in a SRY-negative XX male Podenco dog.

Normal mammalian sex differentiation takes place in three genetically controlled steps: chromosomal sex determination (XX or XY), gonadal differentiation and development of the phenotypic sex. Animals are considered to be sex reversed if chromosomal sex determination and gonadal development are not in agreement. In this report, sex reversal is described in a 1.5-year-old Podenco dog that was referred because of suspected recurrent growth of a previously removed os clitoridis in the vulva. With that exception the dog was phenotypically female, but had never been in oestrus and exhibited male behaviour. Abdominal ultrasonography showed a small tubular structure dorsal to the bladder, consistent with a uterus. An ovoid structure resembling a gonad was visible between the right kidney and inguinal canal. Plasma testosterone concentrations before and after GnRH administration indicated the presence of functional testicular tissue. Two testes, each with its epididymis and ductus deferens, and a complete bicornuate uterus were removed surgically. Cytogenetic analysis of peripheral blood lymphocytes showed a normal female karyotype (78, XX). These findings are consistent with the diagnosis of an XX male. PCR analysis of genomic DNA revealed that the SRY gene was absent. In summary, this report describes the first SRY-negative XX male Podenco dog with an almost complete female phenotype despite high basal and stimulated plasma testosterone concentrations. It is hypothesized that the clinical observations in this dog may have been caused by reduced and delayed Müllerian-inhibiting substance secretion and the absence of conversion of testosterone to dihydrotestosterone due to 5alpha-reductase deficiency.

Absence of Y-chromosome sequences in tumors from African women with AIDS-related Kaposi sarcoma.

Kaposi sarcoma (KS) occurs with relatively high frequency in immunosuppressed transplant recipients and in patients with AIDS. Recently, Italian investigators reported transplant-related KS tumors bearing donor-derived antigens, suggesting possible parenteral transmission of KS as whole cells, i.e., chimeric tumors. To investigate the hypothesis that KS whole cells may also be transmitted into immunocompromised persons via heterosexual acts, we tested nodular KS lesions and matched normal tissue obtained from female patients with AIDS for the presence of the Y-chromosome specific sex determining sequence (SRY). Among 25 unique tumors tested, none was positive for SRY sequence. While our results do not exclude sexual cellular transmission of whole KS cells, they suggest that if it occurs, it is rare.

Comparative analysis of anti-mouse SRY antibodies.

The Y chromosome gene SRY is the initiator of male sexual differentiation in mammals, but the molecular and cellular mechanisms operating downstream of SRY remain undefined. A deeper understanding of these issues relies on the ability to visualize SRY protein endogenously under a number of experimental conditions. Here we compare the specificity and effectiveness of several available antibodies to mouse SRY. Two antibodies cross-reacted with other SOX proteins in immunofluorescence analyses of transfected cells, and one of these two was unable to detect SRY on Western blots. A third antibody was both avid and specific, and was able to detect endogenous SRY in developing Sertoli cells in mouse genital ridges. Our findings underline the need to distinguish between useful and spurious reagents for biochemical and immunolocalization studies involving mouse SRY protein.

[Pure gonadal dysgenesis XX and XY: observations in fifteen patients]. / Dysgénésies gonadiques pures XX et XY: à propos de 15 cas.

BACKGROUND: Pure gondal dysgenesis is characterized by impuberism with a female phenotype without genital ambiguity. The aim of the study is to describe the diagnostic and therapeutic patterns as well as the clinical features. PATIENTS AND METHODS: A retrospective study of 15 patients with pure gonadal dysgenesis (15 patients, 46 XX and two 46 XY). Clinical parameters, familial cases, serum gonadotropin levels, pelvic ultrasonography, endoscopic data, karyotype, analysis of SRY (sex determining Y chromosome) and therapeutic control and clinical course were recorded. RESULTS: Average age at diagnosis was 21+/-2.83 years. Primary amenorrhea was the most frequent reason for consultation. A familial case was found in five patients. The association of sensorineural deafness was noted in one patient, suggesting Perrault's syndrome. Serum gonadotropin levels were elevated. Celioscopic evaluation carried out for six patients confirmed the diagnosis. There was one case of uterine and vaginal aplasia association (Mayer-Rokytansky-Küster-Hauser syndrome). In one XY patient, SRY analysis was normal. Prophylactic gonadectomy was performed in both XY patients. Substitution therapy was initiated in 11 patients. Follow-up in 6 patients revealed development of secondary sexual characters. DISCUSSION: The clinical, biological and histological features of our patients presenting pure gonadal dysgenesis XX were in agreement with earlier reports in the literature. Familial cases suggest possible autosomal transmission. The lack of a mutation in XY patients suggests a post-transcription anomaly. Complete or parital dysgenesis can be identified by histological analysis of the gondads. CONCLUSION: Study of sex determining genes should provide new perspectives for earlier diagnosis and treatment of pure gondadal dysgenesis.

Ontogeny and cellular localization of SRY transcripts in the human testes and its detection in spermatozoa.

The sex-determining region on the Y (SRY) gene is unequivocally designated as the testis-determining factor in mammals; however, its roles beyond sex determination, if any, have been hitherto unknown. To determine whether SRY has any roles beyond sex determination, herein the expression of SRY mRNA was investigated in the midtrimester human fetal, infantile and adult testes as well as in ejaculated spermatozoa. High levels of SRY transcripts were in situ localized to the Sertoli cells of the developing testis at 9 weeks of gestation, and the expression persisted at comparable levels throughout the midtrimester (until 22 weeks) and also in the testis of an infant at 3 months of age. The germ cells and other somatic cells in the testes of fetuses and the infant were negative for SRY expression. The mRNA for SRY was detected in the spermatogenic cells, particularly the spermatogonia and the round spermatids; the expression was negligible in the meiotic stages. A single transcript of approximately 1.2 kb was detected in the adult testes and isolated spermatogonial cells. In the adult testis, in situ hybridization (ISH) studies revealed a switch in the cellular localization of SRY transcripts. SRY transcripts were also demonstrable by RT-PCR of RNA from ejaculated human spermatozoa. ISH revealed the presence of SRY transcripts in the midpiece of 50% of ejaculated sperm. These results suggest that SRY may have extensive roles in male reproductive physiology, such as maturation of fetal testis, spermatogenesis, sperm maturation and early embryonic development.